A. Field of the Invention
Many viral, fungal, bacterial and parasitic diseases are detrimental to human and animal welfare. Convenient, economic and meaningful test procedures are needed to assist the medical and veterinary communities to diagnose and combat these diseases.
This invention relates to a diagnostic method and test kit which have the advantage of being simple to use, economical, rapid and require no special equipment. Since no instruments are required, the test can be run by veterinarians on a farm, by medical and veterinarian personnel in an office setting or in other settings where instruments, running water and other laboratory equipment and supplies are not available.
B. The Prior Art
Many techniques are available to test for microbial diseases. The test of the present invention falls into the category of antibody detection as do many other existing tests. Among these are immunodiffusion, serum neutralization, immunofluorescent staining and the enzyme-linked immunosorbent assay (ELISA) technique developed by Envall and Perlmann in 1972. The samples for these tests are usually blood, plasma, serum or other body fluids or tissues.
All of the tests involve the binding of antibodies in the sample with controlled amounts of antigen. The antigen may either be added in liquid form, as is done with immunodiffusion plates, or immobilized on a surface as part of the test system, as is done with the popular 96-well ELISA technique. Detection of antibody is done by viewing a precipitation line in immunodiffusion techniques, by observing fluorescence in staining techniques, and by observing color generation in enzymatic techniques.
The ELISA multi-well techniques have the following procedural similarities:
1. Wells of polystyrene micro-titer plates are sensitized by passive absorption with the relevant antigen; the plates are then washed.
2. The test samples are incubated in the sensitized well and the plates are again washed. Antibody present in that sample reacts with and is bound to the immobilized antigen on the well surfaces.
3. Enzyme-labeled anti-Ig (i.e., of an immunoglobulin animal species corresponding to the sample) conjugate is incubated in the wells. The conjugate contains an enzyme such as peroxidase, glucose oxidase, beta-galactosidase or alkaline phosphatase. The conjugate reacts with any "captured" or bound antibody. Excess reagent is washed away.
4. Enzyme substrate is added and the plates are incubated; the rate of degradation is indicated by a color change, which is proportional to the antibody concentration in the test samples in Step 2.
5. The reaction is stopped or allowed to arrest and the color change is assessed visually or in a spectrophotometer.
The use of an ELISA-type antibody detection technique to diagnose pseudorabies in swine is exemplified by Joo U.S. Pat. No. 4,562,147.
The basic technique of the ELISA test has been modified by the method of the present invention to significantly reduce the steps needed to conduct the test and to enable the user to read results without the need of instrumentation.